Jochen P. Zubrod

Fungal composition on leaves explains pollutant-mediated indirect effects on amphipod feeding

The energy stored in coarse particulate organic matter, e.g. leaf litter, is released to aquatic ecosystems by breakdown processes involving microorganisms and leaf shredding invertebrates. The palatability of leaves and thus the feeding of shredders on leaf material are highly influenced by microorganisms. However, implications in the colonization of leaves by microorganisms (=conditioning) caused by chemical stressors are rarely studied. Our laboratory experiments, therefore, investigated for the first time effects of a fungicide on the conditioning process of leaf material by means of food-choice experiments using Gammarus fossarum (Crustacea: Amphipoda). Additionally, microbial analyses were conducted to facilitate the mechanistic understanding of the observed behavior. Gammarids significantly preferred control leaf discs over those conditioned in presence of the fungicide tebuconazole at concentrations of 50 and 500 μg/L. Besides the decrease of fungal biomass with increasing fungicide concentration, also the leaf associated fungal community composition showed that species preferred by gammarids, such as Alatospora acumunata, Clavariopsis aquatica, or Flagellospora curvula, were more frequent in the control. Tetracladium marchalianum, however, which is rejected by gammarids, was abundant in all treatments suggesting an increasing importance of this species for the lower leaf palatability--as other more palatable fungal species were almost absent--in the fungicide treatments. Hence, the food-choice behavior of G. fossarum seems to be a suitable indicator for alterations in leaf associated microbial communities, especially fungal species composition, caused by chemical stressors. Finally, this or similar test systems may be a reasonable supplement to the environmental risk assessment of chemicals in order to achieve its protection goals, as on the one hand, indirect effects may occur far below concentrations known to affect gammarids directly, and on the other hand, the observed shifts in leaf associated microbial communities may have perpetuating implications in leaf shredding invertebrates.

Ecotoxicological impact of the fungicide tebuconazole on an aquatic decomposer‐detritivore system

Leaf litter breakdown is a fundamental process in aquatic ecosystems that is realized by microbial decomposers and invertebrate detritivores. Although this process may be adversely affected by fungicides, among other factors, no test design exists to assess combined effects on such decomposer-detritivore systems. Hence, the present study assessed effects of the model fungicide tebuconazole (65 µg/L) on the conditioning of leaf material (by characterizing the associated microbial community) as well as the combined effects (i.e., direct toxicity and food quality-related effects (=indirect)) on the energy processing of the leaf-shredding amphipod Gammarus fossarum using a five-week semistatic test design. Gammarids exposed to tebuconazole produced significantly less feces (≈ 20%), which in turn significantly increased their assimilation (≈ 30%). Moreover, a significantly reduced lipid content (≈ 20%) indicated lower physiological fitness. The conditioning process was altered as well, which was indicated by a significantly reduced fungal biomass (≈ 40%) and sporulation (≈ 30%) associated with the leaf material. These results suggest that tebuconazole affects both components of the investigated decomposer-detritivore system. However, adverse effects on the level of detritivores cannot be explicitly attributed to direct or indirect pathways. Nevertheless, as the endpoints assessed are directly related to leaf litter breakdown and associated energy transfer processes, the protectiveness of environmental risk assessment for this ecosystem function may be more realistically assessed in future studies by using this or comparable test designs.

The functional and physiological status of Gammarus fossarum (Crustacea; Amphipoda) exposed to secondary treated wastewater

Climate change scenarios predict lower flow rates during summer that may lead to higher proportions of wastewater in small and medium sized streams. Moreover, micropollutants (e.g. pharmaceuticals and other contaminants) continuously enter aquatic environments via treated wastewater. However, there is a paucity of knowledge, whether extended exposure to secondary treated wastewater disrupts important ecosystem functions, e.g. leaf breakdown. Therefore, the amphipod shredder Gammarus fossarum was exposed to natural stream water (n=34) and secondary treated wastewater (n=32) for four weeks in a semi-static test system under laboratory conditions. G. fossarum exposed to wastewater showed significant reductions in feeding rate (25%), absolute consumption (35%), food assimilation (50%), dry weight (18%) and lipid content (22%). Thus, high proportions of wastewater in the stream flow may affect both the breakdown rates of leaf material and thus the availability of energy for the aquatic food web as well as the energy budget of G. fossarum.

Effects of subchronic fungicide exposure on the energy processing of Gammarus fossarum (Crustacea; Amphipoda)

Current aquatic environmental risk assessment of plant protection products or biocides does not consider effects on organisms involved in leaf litter breakdown, a fundamental ecosystem process in streams. Therefore, direct ecotoxicological implications of tebuconazole, a frequently used triazole fungicide, on the leaf-shredding amphipod Gammarus fossarum, were assessed. While acute toxicity was low (96h-LC(50)=1347 μg/L), feeding rate, a sublethal endpoint, was significantly reduced after seven days of exposure to 600 μg/L. At the same concentration, but during a three week exposure under semi-static conditions, gammarids showed significant reductions in feeding, but also in assimilation and growth. At 200 μg/L, however, only assimilation was significantly affected. As these endpoints can be used to evaluate the ecotoxicity of a broad range of chemicals and to deduce possible implications in the functioning of ecosystems, the inclusion of similar experimental set-ups might further improve aquatic environmental risk assessment.

Effects of fungicides on decomposer communities and litter decomposition in vineyard streams

Large amounts of fungicides are applied globally and partly enter freshwater ecosystems. A few laboratory studies examined their effects on decomposer communities and the ecosystem process of litter decomposition (LD), whereas the field situation remains largely unknown. We conducted a field study with 17 stream sites in a German vineyard area where fungicides represent the dominant pest control agent. Passive samplers were used to monitor 15 fungicides and 4 insecticides in streams and their toxicity was described using the toxic unit approach, whereas sediment samples were taken to characterise total copper concentrations. Microbial and leaf-shredding invertebrate community composition and related LD rates were assessed at each site. The structure of microbial and shredder communities as well as fungal biomass changed along the fungicide toxicity gradient. The changes in microbial endpoints were associated with a reduction of microbial LD rate of up to 40% in polluted streams. By contrast, neither the invertebrate LD rate nor in-situ measured gammarid feeding rates correlated with fungicide toxicity, but both were negatively associated with sediment copper concentrations. A subsequent laboratory experiment employing field fungicide concentrations suggested that the microbial community changes are causal. Overall, our results suggest that fungicides can affect LD under field conditions.

Effects of nano-TiO2 in combination with ambient UV-irradiation on a leaf shredding amphipod

Production and use of engineered nanoparticles, such as titanium dioxide nanoparticles (nTiO(2)), is increasing worldwide, enhancing their probability to enter aquatic environments. However, direct effects of nTiO(2) as well as ecotoxicological consequences due to the interactions of nTiO(2) with environmental factors like ultraviolet (UV) irradiation on representatives of detrital food webs have not been assessed so far. Hence, the present study displayed for the first time adverse sublethal effects of nTiO(2) at concentrations as low as 0.2 mg L(-1) on the leaf shredding amphipod Gammarus fossarum both in presence and absence of ambient UV-irradiation following a 7-d exposure. In absence of UV-irradiation, however, the effects seemed to be driven by accumulation of nTiO(2) at the bottom of the test vessels to which the gammarids were potentially exposed. The adverse sublethal and lethal effects on gammarids caused by the combined application of nTiO(2) and ambient UV-irradiation are suggested to be driven by the formation of reactive oxygen species. In conclusion, both the accumulation of nTiO(2) at the bottom of the test vessel and the UV induced formation of reactive oxygen species clearly affected its ecotoxicity, which is recommended for consideration in the environmental risk assessment of nanoparticles.

Cryptic species diversity: an overlooked factor in environmental management?

Summary Molecular genetic methods continuously uncover cryptic lineages harboured by various species. However, from an applied perspective, it remains unclear whether and to which extent such a genetic diversity affects biological traits (e.g. ecological, behavioural and physiological characteristics) and environmental management. We assessed potential deviations regarding the trait ‘environmental stress tolerance’ using individuals from five field populations of each of two cryptic lineages (called A and B) comprised under the nominal species Gammarus fossarum. We used ammonia as a chemical stressor while assessing the feeding rate on leaf discs as a measure of sublethal response. In this context, we established a restriction fragment length polymorphism assay to allow a rapid identification of the lineages. We observed a biologically meaningful and statistically significant twofold higher overall tolerance of one cryptic lineage, lineage B, over the other. Confounding factors that may have the potential to influence the test results, such as life stage, sex, season of collection, parasitism, physiological status of organisms and upstream land-use patterns of the river catchments, were either controlled for or displayed only minor deviations between lineages. Synthesis and applications. The trait differences observed in the present study seem to be mainly explained by the considerable genetic differentiation between cryptic lineages of one nominal species. Although traits other than tolerance have been minimally investigated in this context, this study indicates implications in the reliability and quality of environmental monitoring and management if cryptic lineage complexes are ignored.

Ecotoxicological evaluation of three tertiary wastewater treatment techniques via meta-analysis and feeding bioassays using Gammarus fossarum.

Advanced treatment techniques, like ozone, activated carbon and TiO(2) in combination with UV, are proposed to improve removal efficiency of micropollutants during wastewater treatment. In a meta-analysis of peer-reviewed literature, we found significantly reduced overall ecotoxicity of municipal wastewaters treated with either ozone (n=667) or activated carbon (=113), while TiO(2) and UV was not yet assessed. As comparative investigations regarding the detoxification potential of these advanced treatment techniques in municipal wastewater are scarce, we assessed them in four separate Gammarus-feeding trials with 20 replicates per treatment. These bioassays indicate that ozone concentrations of approximately 0.8mg ozone/mg DOC may produce toxic transformation products. However, referred effects are removed if higher ozone concentrations are used (1.3mg ozone/mg DOC). Moreover, the application of 1g TiO(2)/l and ambient UV consistently reduced ecotoxicity. Although activated carbon may remove besides micropollutants also nutrients, which seemed to mask its detoxification potential, this treatment technique reduced the ecotoxicity of the wastewater following its amendment with nutrients. Hence, all three advanced treatment techniques are suitable to reduce the ecotoxicity of municipal wastewater mediated by micropollutants and may hence help to meet the requirements of the European Water Framework Directive.

Inorganic fungicides as routinely applied in organic and conventional agriculture can increase palatability but reduce microbial decomposition of leaf litter

Summary The application of fungicides is considered an indispensable measure to secure crop production. These substances, however, may unintentionally enter surface waters via run-off, potentially affecting the microbial community. To assess such risks adequately, authorities recently called for suitable test designs involving relevant aquatic micro-organisms. We assessed the structural and functional responses of leaf-associated microbial communities, which play a key role in the breakdown of allochthonous leaf material in streams, towards the inorganic fungicides copper (Cu) and elemental sulphur (S). These substances are of particular interest as they are authorized for both conventional and organic farming in many countries of the world. We used the food choice of the amphipod shredder Gammarus fossarum (indicative for micro-organism-mediated leaf palatability) as well as microbial leaf decomposition as functional endpoints. Moreover, the leaf-associated microbial communities were characterized by means of bacterial density, fungal biomass and community composition facilitating mechanistic understanding of the observed functional effects. While Gammarus preferred Cu-exposed leaves over unexposed ones, microbial leaf decomposition was reduced by both Cu and S (up to 30%). Furthermore, Cu exposure decreased bacterial densities (up to 60%), stimulated the growth of leaf-associated fungi (up to 100%) and altered fungal community composition, while S did not affect any of the assessed structural endpoints. Synthesis and applications. We observed both structural and functional changes in leaf-associated microbial communities at inorganic fungicide concentrations realistic for surface water bodies influenced by conventional and organic farming. Our data hence justify a careful re-evaluation of the environmental safety of the agricultural use of these compounds. Moreover, inclusion of an experimental design similar to the one used in this study in lower tier environmental risk assessments of antimicrobial compounds may aid to safeguard the integrity of aquatic microbial communities and the functions they provide.

The relative importance of diet-related and waterborne effects of copper for a leaf-shredding invertebrate

Copper (Cu) exposure can increase leaf-associated fungal biomass, an important food component for leaf-shredding macroinvertebrates. To test if this positive nutritional effect supports the physiological fitness of these animals and to assess its importance compared to waterborne toxicity, we performed a 24-day-bioassay in combination with a 2×2 factorial design using the amphipod shredder Gammarus fossarum and a field-relevant Cu concentration of 25 μg/L (n = 65). Waterborne toxicity was negligible, while gammarids fed leaves exposed to Cu during microbial colonization exhibited a near-significant impairment in growth (∼30%) and a significantly reduced lipid content (∼20%). These effects appear to be governed by dietary uptake of Cu, which accumulated in leaves as well as gammarids and likely overrode the positive nutritional effect of the increased fungal biomass. Our results suggest that for adsorptive freshwater contaminants dietary uptake should be evaluated already during the registration process to safeguard the integrity of detritus-based ecosystems.